Knowledge of the biochemical pathways by which cells detect and respond to stimuli is important for the discovery, development, and correct application of pharmaceutical products. Cellular physiology involves multiple pathways, which have complex relationships. For example, pathways split and join; there are redundancies in performing specific actions; and response to a change in one pathway can modify the activity of another pathway, both within and between cells. In order to understand how a candidate agent is acting and whether it will have the desired effect, the end result, and effect on pathways of interest is as important as knowing the target protein.
BioMAP® methods of analysis for determining the pathways affected by an agent or genotype modification in a cell, and for identifying common modes of operation between agents and genotype modifications, are described in International Patent application WO01/067103. Cells capable of responding to factors, simulating a state of interest are employed. Preferably the cells are primary cells in biologically relevant contexts. A sufficient number of factors are employed to involve a plurality of pathways and a sufficient number of parameters are selected to provide an informative dataset. The data resulting from the assays can be processed to provide robust comparisons between different environments and agents.
Among physiological states of interest are immune disorders. Allergy, or hypersensitivity, of the immune system in its different forms affects more than 20% of the human population. Furthermore, man is a highly susceptible species to anaphylaxis. After sensitization with an allergen, a second exposure elicits constriction of the bronchioles, in some cases resulting in death from asphyxia. This allergic reaction is mediated by allergen-specific antibodies, mostly of the IgE class. The antibodies can be directed against a variety of antigens, such as molecules from pollen, fungi, food, house dust mite, hymenoptera venoms or animal danders. The aggregation of mast cell and basophil high-affinity IgE receptors by IgE and antigen causes the release of mediators and cytokines, including histamine, heparin, eosinophil and neutrophil chemotactic factors, leukotrienes and thromboxanes. While an understanding of the inflammatory process in allergic reactions and asthma has improved remarkably over the past decade; ability to control them remains modest. The prevalence of asthma in industrialized countries has increased by almost 80% since 1980. The specific causes for this increase in prevalence are not clear, but the rise in prevalence may be due in part to the absence of effective therapies that reverse the progression of, or cure, this disease.
It is now generally thought that asthma is a syndrome, typically characterized by the three cardinal features of intermittent and reversible airway obstruction, airway hyperresponsiveness, and airway inflammation, that may arise as a result of interactions between multiple genetic and environmental factors. Nevertheless, most cases of the disorder (the so-called “atopic” or “allergic” asthma) occur in subjects whom also exhibit immediate hypersensitivity responses to defined environmental allergens, and challenge of the airways of these subjects with such allergens can produce reversible airway obstruction. It is also known that the overall incidence of asthma in several different populations exhibits a strong positive correlation with serum concentrations of IgE, which, in humans, is the main Ig isotype that can mediate immediate hypersensitivity responses.
Mast cells, derivatives of hematopoietic precursor cells that undergo their terminal stages of differentiation/maturation in the peripheral tissues in which they reside, express cell surface receptors (FcRI) that permit them to bind the Fc portion of IgE with high affinity. Such IgE-sensitized mast cells, upon encounter with specific antigen that is recognized by their FcRI-bound IgE, secrete a broad panel of bioactive mediators, including: preformed mediators that are stored in the cell's cytoplasmic granules, e.g. histamine, heparin, and neutral proteases, newly synthesized lipid products, e.g. prostaglandin D2 and leukotriene C4, and diverse cytokines. Many of these potentially mast cell-derived mediators can promote reversible airway obstruction, bronchial hyperreactivity, and/or airway inflammation.
However, additional cell types, including eosinophils and Th2 lymphocytes, both of which are well represented in the chronic inflammatory infiltrates in the airways of patients with asthma, also can produce cytokines or other mediators that may contribute to many of the features of the disease. The FcRI, which was once thought to be restricted to tissue mast cells and basophils, is also expressed on the surface of monocytes, circulating dendritic cells, Langerhans' cells, and eosinophils, thus implicating these cells as additional potential sources of mediators in various IgE-dependent inflammatory responses. (For a review, see Galli (1997) J.E.M. 186:343-347, which disclosure and the references cited therein are herein incorporated by reference)
Current evidence supports a complex view of the pathogenesis of allergic diseases, in which both T cells and mast cells (and other FcRI+ cells) can have both effector cell and immunoregulatory roles in these disorders. This indicates that therapeutic approaches for asthma and other allergic diseases must consider the effect on multiple systems. For example, anti-IgE-based strategies not only may reduce CD23-dependent antigen presentation and FcRI+ cell effector function, but also may diminish FcRI+ cell immunoregulatory function by reducing both mast cell (or basophil) IL-4/IL-13 production and FcRI+-dependent antigen presentation. Conversely, the findings that corticosteroids and other immunosuppressive drugs can diminish mast cell cytokine production, as well as reduce IgE- and mast cell-dependent inflammation and leukocyte recruitment in vivo, raise the possibility that the clinical benefits of such agents in asthma may reflect, at least in part, actions on mast cells as well as on the T cells, eosinophils, and other effector and target cells that participate in these complex disorders.
Given the large number of cells and pathways involved in the development of atopic disease, the evaluation of therapeutic modalities requires a complex assessment of effectiveness in multiple systems. The present invention addresses these issues.